Fig 1.
Proposed progesterone metabolism in human endometrium.
Progesterone is metabolized by 20-ketosteroid reductases and 5α-reductases, to form (20S/R)-pregn-4-ene-3α,20-diol and 5α-pregnane-3,20-dione. Reductions of the 3-keto and 20-keto groups are catalysed by the aldo-keto reductases AKR1C1-AKR1C3. Here, AKR1C1-AKR1C3 can act on the 20-keto group of pregn-4-enes, and they can reduce both 3-keto and 20-keto groups of 5α-pregnanes. The progesterone (20S)-pregn-4-ene-3α,20-diol formed by the AKR1C enzymes can be oxidized back to progesterone by the 20α-hydroxysteroid dehydrogenase activity of 17β-hydroxysteroid dehydrogenase type 2 (HSD17B2). The formation of 5α-pregnanes is irreversible and is catalysed by 5α-reductases types 1 (SRD5A1) and 2 (SRD5A2) [35–37].
Table 1.
Recovery calculations (n = 5) before addition of individual steroids.
Fig 2.
Reaction scheme and structural formulae of the pregn-4-ene-metabolites.
Reagents and conditions: (a) Al(O-i-Pr)3, 2-propanol, reflux.
Table 2.
Selected reaction monitoring transitions and the optimum LC-MS/MS conditions for the internal standard testosterone, progesterone and its metabolites.
Fig 3.
Representative chromatogram showing separation of progesterone and its metabolites.
Representative extracted- ion chromatogram of a 100 ng/mL standard mixture of progesterone and its metabolites.
Fig 4.
Representative calibration curves in solvent.
A) (20S)-pregn-4-ene-3α,20-diol (r = 0.9999, y = 0.00015x). B) (20S)-5α-pregnane-3α,20-diol (r = 0.9998, y = 0.00014). Curves are constructed out of three measurements for each concentration.
Fig 5.
Representative calibration curves in matrix.
A) (20S)-pregn-4-ene-3α,20-diol (r = 0.9985, y = 0.00015x). B) (20S)-5α-pregnane-3α,20-diol ((r = 0.9997, y = 0.00015). Curves are constructed out of three measurements for each concentration.
Table 3.
Recovery calculations (n = 5) after addition of 10 ng/mL of individual steroid.
Table 4.
Recovery calculations (n = 5) after addition of 50 ng/mL of individual steroid.
Table 5.
Recovery calculations (n = 5) after addition of 250 ng/mL of individual steroid.
Fig 6.
Progesterone metabolites formed in the HES cell line.
Histogram representing the percentages of progesterone and its most abundant metabolites detected at different times. The scheme of the proposed metabolism is shown below. The main pathway is marked in bold. First, the 20-keto group of progesterone is reduced to form (20S)-20-hydroxy-pregn-4-ene-3-one, which is reduced at position C5 to form (20S)-20-hydroxy-5α-pregnane-3-one. Then (20S)-20-hydroxy-5α-pregnane-3-one is further metabolized at the 3-keto group, to form mainly (20S)-5α-pregnane-3α,20-diol, and also (20S)-5α-pregnane-3β,20-diol to a lesser extent.